The project is directed toward identifying the actual products of chemical reactions between mutagens and carcinogens and informational macromolecules. Specifically, we propose to study the effect of alkylating agents on polynucleotides and nucleic acids in vitro and in vivo. We propose to continue our present work on the ethylation of nucleotides, RNA, and DNA in vitro and animal cells in vivo by diethylsulfate, a poor mutagen, and ethyl methanesulfonate, a good mutagen, in order to identify and quantitate all alkyl bases formed, particularly at very low levels of alkylation. Both the specific reagent used and the conformation of the nucleic acid play an important role in determining the preferred site of a major extent only when the polynucleotide is unstacked and that only under these conditions is TMV- RNA mutated. This modification was further shown to be mutagenic when 3-methyl cytidine, incorporated into polycytidylic acid, caused random incorporation of all triphosphates when used as template by RNA polymerase. Our more recent experiments indicate that amino ethylation of cytidine and adenosine, 6-0 and 3-ethylation of guanosine and phosphodiester ethylation do occur and can be detected. We intend to study whether or not these or other modifications are mutagenic or carcinogenic by reacting biologically active nucleic acids, such as transforming DNA of B. subtilis, phi X174 DNA, and TMV-RNA with several differing alkylating agents (e.g., diethyl sulfate, ethyl methanesulfonate, N-ethyl nitrosourea or diethylnitrosamine) under conditions of pH, solvent, or time which favor one or the other of these reactions and correlate the chemical analysis with the extent of mutation.